Supplementary materials for
Analysis of the complete genome sequence of a marine streptomycete Streptomyces sp. S063 CGMCC 14582 reveals its potential to produce novel secondary metabolites including anti-complement agents
Liang-Yu Chen1, Hao-Tian Cui2, Chun Su3*, Feng-Wu Bai2 and Xin-Qing Zhao2*
1School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116023, China.
2State Key Laboratory of Microbial Metabolism and School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
3National Engineering Laboratory for Resource Developing of Endangered Chinese Crude Drugs in Northwest China, College of Life Sciences, Shaanxi Normal University, Xi’an 710119, China.
*Corresponding authors:
Dr. Chun Su. E-mail: [email protected]. Tel: +86-29-85310581. Fax: +86-29-85310623; Prof. Dr. Xinqing Zhao, E-mail: [email protected]. Tel: +86-21-34206673. Fax: +86-21-34208028.
Supplementary tables
Table S1 Culture medium used in this study
Name
Composition (g/L)
pH
A1
yeast extract 4, soluble starch 10, peptone 2, sea salt 28
7.0
TSB
tryptone 17, soy peptone 3, glucose 2.5, K2HPO4 2.5
7.2
MS
starch or cellulose 10, K2HPO4 0.5, MgSO4 0.5, NaCl 0.5,KNO3 1, agar 20
7.0
M2
soluble starch 20, glucose 10, peptone 5, yeast extract 5, CaCO3 5
7.0
M3
soluble starch 20, soybean powder 25, NH4Cl 2, NaCl 2, K2HPO4 0.5, CaCO3 5
7.2
M8
malt extract 40, yeast extract 4, glucose 2, NH4Cl 0.8, Na2HPO4 1
6.0
M9
sucrose 20, soybean powder 10, corn syrup 10, KCl 8
6.5
M12
glucose 100, yeast extract 10, CaCO3 2
6.8
M17
glucose 10, soy flour 10, corn syrup 10, glycerol 5, yeast extract 5, NaCl 5, CaCO3 2
5.7
M19
soluble starch 20, cotton seed powder 5, yeast extract 2.5, NaCl 1, K2HPO4 0.75, MgSO4·7H2O 1, CaCO3 3
7.5
M22
malt extract 35, corn starch 30, corn syrup 15, cotton seed powder 15, CaCO3 2
7.5
Table S2 Primers used in this study
Name
Sequence
apra F
TGTAGGCTGGAGCTGCTTC
apra R
TATTCCGGGGATCCGTCGAC
4-k1 F
GCCTGCAGGTCGACTCTAGA CGACGGAAACACGGGAGAATCA
4-k1 R
CGAAGCAGCTCCAGCCTACA TGAAGTGCTCCACGAACAGTCC
4-k3 F
TCGACGGATCCCCGGAATAT TACGGAGTCGTGACGTGTGC
4-k3 R
AATTCGAGCTCGGTACCCGG CGGAGCCGTGAACTGACACT
23-k1 F
GCCTGCAGGTCGACTCTAGA CGCAAGTACCACAAGCACCTGA
23-k1 R
CGAAGCAGCTCCAGCCTACA TGTCGGAGTTCCTGGGCTGA
23-k3 F
TCGACGGATCCCCGGAATAT AGATGCTGCTGCGCACTCT
23-k3 R
AATTCGAGCTCGGTACCCGG CTACCAGACCACCCGGAGC
25-k1 F
GCCTGCAGGTCGACTCTAGA GCACGGATCGCCGAATGGAT
25-k1 R
CGAAGCAGCTCCAGCCTACA TGGTGGGCCAGCATCTCGAT
25-k3 F
TCGACGGATCCCCGGAATAT GCGGTGATCTCATCGATTCGG
25-k3 R
AATTCGAGCTCGGTACCCGG GGGGCTCATGAACCGGCG
28-k1 F
GCCTGCAGGTCGACTCTAGA ATGAGCACCCAGACAGCCCA
28-k1 R
CGAAGCAGCTCCAGCCTACA TTCAGGCCTGCTGGTGCAG
28-k3 F
TCGACGGATCCCCGGAATAT GGTCAGCCGTTGAACGTGTCC
28-k3 R
AATTCGAGCTCGGTACCCGG ATGCGCATGCGACCCACC
4-773 F
ACGACGTCCTCGCCACCGGACTGTTCGTGGAGCACTTCA TGTAGGCTGGAGCTGCTTC
4-773 R
GACGGCCGGGGTGCGGCGCGCACACGTCACGACTCCGTA TATTCCGGGGATCCGTCGAC
4-p18 F
ACCGAACGACCGGGTGGCCAGTGTCAGTTCACGGCTCCG CCGGGTACCGAGCTCGAATT
4-p18 R
CTCTCTCGGTTGTCGAATGATTCTCCCGTGTTTCCGTCG TCTAGAGTCGACCTGCAGGC
23-773 F
AGCCCCTTCGGGTCGCGGGTCAGCCCAGGAACTCCGACA TGTAGGCTGGAGCTGCTTC
23-773 R
AGGGGTCGACGGAGACGTACAGAGTGCGCAGCAGCATCT TATTCCGGGGATCCGTCGAC
23-p18 F
ACCTCCCGCAGCCCGCGGCGGCTCCGGGTGGTCTGGTAG CCGGGTACCGAGCTCGAATT
23-p18 R
GGACATCTCCGACAGCTTCAGGTGCTTGTGGTACTTGCG TCTAGAGTCGACCTGCAGGC
25-773 F
AGAGGCGCTGGAAGGCCTGATCGAGATGCTGGCCCACCA TGTAGGCTGGAGCTGCTTC
25-773 R
GTGGGGACGGCAGGCCGTCCGAATCGATGAGATCACCGC TATTCCGGGGATCCGTCGAC
25-p18 F
CATCACCCCCGCGGCCCCGCCCGCCGGTTCATGAGCCCC CCGGGTACCGAGCTCGAATT
25-p18 R
CGCCGGTGACGTCGCCGCGATCCATTCGGCGATCCGTGC TCTAGAGTCGACCTGCAGGC
28-773 F
ATGCGGTGGACCCCCGCACCCTGCACCAGCAGGCCTGAA TGTAGGCTGGAGCTGCTTC
28-773 R
GCGCACCGGGCCCGACCCGGACACGTTCAACGGCTGACC TATTCCGGGGATCCGTCGAC
28-p18 F
CTCGGCGGAGGTCCAGCTCAGGGTGGGTCGCATGCGCAT CCGGGTACCGAGCTCGAATT
28-p18 R
GGCGGTCGGTGCCTCGCGGTGGGCTGTCTGGGTGCTCAT TCTAGAGTCGACCTGCAGGC
4yan F
ACACGATCATCGACCACACCTG
23yan F
CATCGAGGTCGAGCACCTGTT
25yan F
TACGGCAAGCCGTACTGGA
28yan F
CGTGATCGAACTGCTGCGC
773yan R
ATCCATTGCCCCTGCCACCT
Note: The underlined letters indicate the homologous regions.
Table S3 General features of Streptomyces sp. S063
Items
Description
Classification
Domain Bacteria
Phylum Actinobacteria
Class Actinobacteria
Order Actinomycetales
Family Streptomycetaceae
Genus Streptomyces
Species Streptomyces badius
Strain: S063
Gram stain
Positive
Motility
Motionless
Pigmentation
Yellowish white
Cell shape
Branched mycelia
Temperature range (°C)
4-37, optimally at 28
Biotic relationship
Free living
Salinity (w/v)
0.5-9.5%, optimum 3.5% NaCl*
Relation to oxygen
Aerobic
Note: *, TSB medium contains 0.5% (w/v) NaCl.
Table S4 Number of genes associated with general COG functional categories
Code
No.
Percentage
Description
J
208
2.93
Translation, ribosomal structure and biogenesis
A
4
0.06
RNA processing and modification
K
683
9.61
Transcription
L
233
3.28
Replication, recombination and repair
B
2
0.03
Chromatin structure and dynamics
D
53
0.75
Cell cycle control, Cell division, chromosome partitioning
V
129
1.81
Defense mechanisms
T
429
6.03
Signal transduction mechanisms
M
269
3.78
Cell wall/membrane biogenesis
N
8
0.11
Cell motility
U
46
0.65
Intracellular trafficking and secretion
O
159
2.24
Posttranslational modification, protein turnover, chaperones
C
294
4.14
Energy production and conversion
G
456
6.50
Carbohydrate transport and metabolism
E
462
6.54
Amino acid transport and metabolism
F
112
1.58
Nucleotide transport and metabolism
H
231
3.25
Coenzyme transport and metabolism
I
264
3.71
Lipid transport and metabolism
P
251
3.53
Inorganic ion transport and metabolism
Q
243
3.42
Secondary metabolites biosynthesis, transport and catabolism
R
753
10.59
General function prediction only
S
423
5.95
Function unknown
-
2,740
38.54
Not in COGs
Note: The percentage is calculated based on the total number of protein coding genes in the genome.
Table S5 Genome comparison of Streptomyces sp. S063 and the subspecies of S. griseus using OrthoANIu values Organism/NameStrainAssemblySize (Mb)ScaffoldsGeneOrthoANIu value (%)AAL (bp) #
S. griseus subsp. griseusNBRC 13350GCA_000010605.18.55 1*7,28489.94,055,388
S. griseusBIG105GCA_001715295.16.87 16**5,99682.452,763,591
S. griseusNRRL B-2165GCA_001723115.18.10 1,108**7,27790.023,607,713
S. griseus subsp. griseusNRRL B-2682GCA_001723125.17.96 1,244**7,20290.223,539,928
S. griseusS4-7GCA_000932225.27.69 36,92793.534,320,872
S. griseusDSM 40236GCA_900105705.18.63 47,38189.874,138,770
S. griseus subsp. rhodochrousNRRL B-2931GCA_000718205.18.03 677,12088.73,818,413
S. griseus subsp. rhodochrousNRRL B-2930GCA_000721205.18.03 857,11488.793,794,648
S. griseus subsp. griseusNRRL F-2227GCA_000721575.18.04 867,05588.753,829,922
S. griseus subsp. griseusNRRL F-5144GCA_000719355.17.85 897,11093.64,267,182
S. griseus subsp. rhodochrousNRRL B-2929GCA_001270675.18.03 1027,12588.793,802,556
S. griseus subsp. griseusNRRL B-2307GCA_000717375.17.20 1356,39178.822,288,652
S. griseus subsp. rhodochrousNRRL B-2932GCA_000718235.17.84 1426,80888.863,844,738
S. griseus subsp. griseusNRRL F-5618GCA_000719435.17.10 1656,24778.822,349,132
S. griseus subsp. griseusNRRL WC-3480GCA_000717795.18.33 1667,17789.94,021,963
S. griseus subsp. griseusNRRL B-2621GCA_000721035.17.43 1746,71779.432,402,716
S. griseus subsp. rhodochrousNRRL B-1691GCA_000719035.18.09 2287,12488.753,798,650
S. griseus subsp. griseusNRRL B-2621GCA_000718525.17.43 2396,73179.492,414,674
S. griseus subsp. griseusNRRL F-5621GCA_000719495.17.34 2406,43878.752,257,522
S. griseus subsp. griseusNRRL WC-3645GCA_000720255.19.89 2659,04778.142,290,396
S. griseus subsp. griseusNRRL WC-3066GCA_001509565.17.15 2876,32278.822,288,023
S. griseus subsp. rhodochrousNRRL B-2933GCA_000717555.27.84 4,281-89.082,825,857
Note: #AAL, Average aligned length; *, complete level; **, scaffold level; the rest genomes are contig level. OrthoANIu value and AAL value are calculated by comparing Streptomyces sp. S063 with the selected strains.
Table S6 Deduced gene functions of Cluster17
Gene
Identity
Coverage
Subject gene
Function
gene4741
85
61.8
BAG19226.1
AmfT protein
gene4742
100
100.0
BAG19225.1
AmfS protein
gene4743
83
95.8
BAG19224.1
membrane translocator
gene4744
86
97.1
BAG19223.1
membrane translocator
gene4745
92
100.0
BAG19222.1
transcriptional regulator
Table S7 Deduced gene functions of Cluster8
Gene
Identity
Coverage
Subject gene
Function
gene975
98
100.0
EFE73293.1
transmembrane-transport protein
gene976
98
98.4
EFE73294.1
TetR-family transcriptional regulator
gene977
94
100.0
EFE73295.1
conserved hypothetical protein
gene978
98
100.0
EFE73296.1
oxidoreductase
gene979
99
100.0
EFE73297.1
argininosuccinate lyase ArgH
gene980
99
100.0
EFE73298.1
argininosuccinate synthase ArgG
gene981
97
100.0
EFE73300.1
secreted protein
gene982
100
100.0
EFE73301.1
arginine repressor
gene983
96
99.0
EFE73302.1
N2-acetyl-L-ornithine: 2-oxoglutarate aminotransferase ArgD
gene984
97
100.0
EFE73303.1
N-acetylglutamate kinase ArgB
gene985
96
100.0
EFE73304.1
N2-acetyl-L-ornithine: L-glutamate N-acetyltransferase ArgJ
gene986
97
99.7
EFE73305.1
N-acetyl-gamma-glutamylphosphate reductase
gene987
98
100.0
EFE73306.1
cysteine synthase
gene988
95
98.6
EFE73307.1
ornithine cyclodeaminase
gene989
91
101.2
EFE73308.1
non-ribosomal peptide synthetase
gene990
90
100.0
EFE73309.1
conserved hypothetical protein
gene991
91
100.0
EFE73310.1
conserved hypothetical protein
gene992
96
88.0
EFE73312.1
nonribosomal peptide synthetase
gene993
93
94.8
EFE73312.1
nonribosomal peptide synthetase
gene994
92
87.6
EFE73312.1
nonribosomal peptide synthetase
gene995
91
100.0
EFE73312.1
nonribosomal peptide synthetase
gene996
92
96.2
EFE73312.1
nonribosomal peptide synthetase
gene997
88
97.1
EFE73313.1
nonribosomal peptide synthetase
gene998
92
100.4
EFE73313.1
nonribosomal peptide synthetase
gene999
94
97.7
EFE73314.1
pyridoxal-5'-phosphate-dependent enzyme
Table S8 Deduced gene functions of Cluster20
Gene
Identity
Coverage
Subject gene
function
gene5506
53
101.6
BAP34706.1
LuxR-family transcriptional regulator
gene5507
77
96.6
BAP34705.1
NAD-dependent epimerase/dehydratase
gene5508
73
94.7
BAP34704.1
UDP-glucose/GDP-mannose dehydrogenase
gene5509
64
96.9
BAP34703.1
LmbE family protein
gene5510
62
103.4
BAP34763.1
type I polyketide synthase
gene5515
63
106.0
BAP34740.1
type I polyketide synthase
gene5516
62
89.1
BAP34739.1
type I polyketide synthase
gene5517
59
105.5
BAP34738.1
regulatory protein TetR
gene5518
72
94.9
BAP34737.1
ABC-type multidrug transport system
gene5519
68
100.0
BAP34736.1
ABC-type multidrug transport system
gene5520
60
97.7
BAP34735.1
thioesterase involved in non-ribosomal peptide biosynthesis
gene5521
61
101.9
BAP34734.1
type I polyketide synthase
gene5522
64
91.6
BAP34733.1
type I polyketide synthase
gene5523
58
97.5
BAP34733.1
type I polyketide synthase
gene5524
59
100.0
BAP34733.1
type I polyketide synthase
gene5525
81
100.8
BAP34719.1
cytochrome P450
gene5526
66
93.9
BAP34718.1
AMP-dependent synthetase and ligase
gene5527
58
100.0
BAP34717.1
acyl carrier protein
gene5528
70
94.3
BAP34707.1
AMP-dependent synthetase and ligase
gene5529
68
94.7
BAP34708.1
malonyl-CoA-ACP transacylase
gene5530
73
97.3
BAP34709.1
(PLP)-dependent aspartate aminotransferase superfamily
gene5531
75
97.3
BAP34710.1
radical-SAM L-lysine 2,3-aminomutase
gene5532
75
98.7
BAP34711.1
proline iminopeptidase
gene5533
77
98.4
BAP34712.1
glycosyltransferase
gene5534
64
99.7
BAP34715.1
glucose-1-phosphate thymidylyltransferase
gene5535
66
91.7
BAP34714.1
dTDP-glucose 4,6-dehydratase
gene5536
48
98.7
BAP34742.1
NDP-hexose 2,3-dehydratase
gene5537
59
97.5
BAP34716.1
N-methyltransferase
gene5538
46
84.8
BAP34741.1
glycosyltransferase
gene5540
46
99.3
BAP34747.1
cytochrome P450
gene5541
61
98.8
BAP34746.1
glycosyltransferase
gene5542
68
96.2
BAP34743.1
NDP-hexose-3-ketoreductase
gene5543
73
98.5
BAP34745.1
aminotransferase
Table S9 Analysis of transporters according to TransportDB transporter family in the genomes of Streptomyces sp. S063 and its related strains
Family
063
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
SSS
1
3
2
4
2
2
2
3
2
7
6
6
2
0
3
0
0
1
0
1
1
1
Nramp
1
3
2
1
1
1
1
1
1
5
4
4
5
2
3
1
2
4
1
1
2
1
TRAP-T
0
1
2
1
1
1
1
1
1
4
4
4
4
4
4
1
1
1
1
1
1
1
BCCT
12
4
4
1
2
3
3
3
3
3
1
1
1
1
2
1
2
2
2
2
2
1
Trk
3
1
1
1
1
2
2
3
3
1
1
1
1
1
3
1
3
3
1
1
1
1
Tat
3
2
2
1
5
5
5
2
2
3
3
3
1
1
1
2
2
2
0
0
0
0
MOP
5
3
3
1
2
4
4
1
1
5
3
3
1
0
2
2
0
3
1
0
0
0
NSS
0
1
1
0
1
1
1
1
1
1
0
0
0
0
0
1
1
1
0
1
0
0
RhtB
1
0
1
1
3
2
2
1
1
1
1
1
0
0
1
0
1
0
0
0
0
0
ArsB
0
0
0
0
0
1
1
0
0
1
1
1
1
0
0
0
0
0
0
0
0
0
ThrE
1
1
1
0
1
1
1
0
0
1
1
1
0
0
0
0
0
0
0
0
0
0
LIV-E
0
0
0
0
1
1
1
0
0
1
1
1
0
0
0
0
0
0
0
0
0
0
DAACS
0
0
0
0
1
1
1
0
0
1
1
1
0
0
0
0
0
0
0
0
0
0
LctP
0
0
0
0
1
1
1
0
0
2
1
1
0
0
0
0
0
0
0
0
0
0
MscL
2
0
0
0
2
1
1
0
0
1
1
1
0
0
0
0
0
0
0
0
0
0
GPTS
2
0
1
0
2
2
1
0
0
2
2
2
0
0
0
0
0
0
0
0
0
0
ACR3
1
0
0
0
1
1
1
0
0
1
1
1
0
0
0
0
0
0
0
0
0
0
NhaA
10
0
0
0
3
3
3
0
0
2
0
0
0
0
0
1
0
0
0
0
0
0
NCS1
0
0
1
6
4
2
2
2
2
3
1
1
1
0
0
0
0
0
0
0
1
0
POT
0
0
0
0
2
2
2
0
0
3
1
1
0
0
0
0
0
0
0
0
0
0
MscS
1
0
0
0
3
3
2
0
0
4
2
2
0
0
0
0
0
0
1
1
0
0
Amt
0
0
0
0
3
3
3
0
0
2
2
2
0
0
0
0
0
0
0
0
0
0
TTT
0
0
0
1
4
2
2
0
0
5
2
3
1
1
1
1
1
2
0
0
0
0
GntP
2
0
0
0
2
2
2
0
0
5
3
3
0
0
0
0
0
0
0
0
0
0
Note: Marine isolated strain was marked in light blue; abundance color of the marine adaption genes: blue, 0; yellow, middle; red, 10 or more.
Strain description: 063, Streptomyces sp. S063; 1, S. sulphureus DSM 40104; 2, S. sulphureus L180; 3, Streptomyces sp. CNT360; 4, S. nanshensis 10399; 5, S. qinglanensis 10379; 6, S. nanshensis 10374; 7, Streptomyces sp. AA1529; 8, Streptomyces sp. CNT318; 9, S. nanshensis 01066; 10, S. abyssalis 10389; 11, S. abyssalis 10390; 12, Streptomyces sp. TAA486; 13, S. nanshensis 10429; 14, Streptomyces sp. TAA204; 15, Streptomyces sp. CNH287; 16, S. oceani 02100; 17, Streptomyces sp. CNS606; 18, Streptomyces sp. W007; 19, Streptomyces sp. CNB091; 20, S. griseus NBRC 13350; 21, S. fulvissimus DSM 40593.
MAT family: SSS, sodium symporter; Nramp, metal ion (Mn2+) transporter; TRAP-T, tripartite ATP-independent periplasmic transporters; BCCT, betaine/carnitine/choline transporter; Trk, K+ transporter; Tat, twin arginine targeting; MOP, multidrug/oligosaccharidyl-lipid/polysaccharides; NSS, neurotransmitter: sodium symporter; RhtB, resistance to homoserine/threonine; ArsB, arsenite-antimonite; ThrE, threonine/serine exporter; LIV-E, branched chain amino acid exporter; DAACS, dicarboxylate/amino acid: cation (Na+ or H+) symporter; LctP, lactate permease; MscL, large conductance mechanosensitive ion channel; GPTS, general phosphotransferase system; ACR3, arsenical resistance-3; NhaA, Na+: H+ antiporters; NCS1, the nucleobase: cation symporter-1; POT, proton-dependent oligopeptide transporter; MscS, small conductance mechanosensitive ion channel; Amt, ammonia transporter channel; TTT, tripartite tricarboxylate transporters; GntP, gluconate: H+ symporter.
Supplementary figure legendsFigure S1 Comparison of the genome of Streptomyces sp. S063 and the reference genomes. Circles from inside to outside: Circle 1, gene location; Circle 2, GC Content; Circle 3, GC Skew; Circle 4-11, genome of S. griseus NBRC 13350, S. fulvissimus DSM 40593, Streptomyces sp. SirexAA-E, S. venezuelae ATCC 10712, S. avermitillis MA-4680, S. albus J1074, S. scabiei 87-22, S. bingchenggensis BCW-1; Circle 12-15, genome of marine-derived strains, Streptomyces sp. CNQ-509, Streptomyces sp. SCSIO 03032, Streptomyces sp. PVA 94-07, Streptomyces sp. PVA 94-10. This comparison figure was built by BLAST Ring Image Generator (BRIG) version 0.95. Figure S2 Neighbor-joining tree showing the phylogenetic relationships of Streptomyces sp. S063 with the related reference strains based on 16S-23S rRNA internal transcribed spacer sequences.
The sequences were selected based on the alignment from BLAST and the NJTree was constructed by Geneious. Bootstrap values were set as 1000 replicates. Bar, 0.01 substitutions per nucleotide position.
Figure S3 The GNPS map of Streptomyces sp. S063.The culture collected from A1 agar was cut into small pieces and extracted by MeOH:Water (1:1), ACN:EtOAc (1:1) and EtOAc, respectively. The samples were analyzed by HPLC-MS/MS and the acquired data was upload and compared with GNPS. The generated data from GNPS was visualized by Cytoscape v3.4.
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Figure S2
Figure S3